Collagen matrix

ABSTRACT

Methods for making a biodegradable collagen matrix having increased osteoinductivity and a biodegradable collagen matrix prepared by these methods are provided. In various embodiments, the methods include providing an acidic collagen slurry and mixing it with at least one water soluble and/or hydrophilic bioactive agent under conditions sufficient to cause the collagen slurry to self-assemble into macroscopic collagen fibers and cause the at least one bioactive agent to form a collagen matrix containing the bioactive agent. Conditions sufficient to cause the collagen slurry to self-assembly include raising the pH of the slurry to from about 5 to about a pH of 9 and/or adding bone powder, calcium phosphate, hydroxyapatite, DBM or a mixture thereof to the acidic collagen slurry in order to raise the pH from about 5 to about 9.

BACKGROUND

Many effective growth factors and other bioactive agents have beendeveloped to help in tissue repair and in treating diseases by applyingthem to a localized area, such as a surgical site. Conventional methodsof delivering growth factors and/or other bioactive agents utilize manytypes of drug delivery technologies.

Current drug delivery technologies utilize different carriers such aspolymeric matrices, microencapsulation, or collagen sponges. In general,polymeric matrix or microencapsulation techniques require theincorporation of the bioactive agent into a carrier at a productionfacility. While release characteristics can be usually controlled,generally the materials used for carriers fail to promote tissue repairand growth.

Collagen sponges are frequently used as scaffolds for tissue growth, butnormally the bioactive agent is added to the sponge at the point of use.In nature, a bioactive agent such as bone morphogenic proteins are foundbound to collagen. Although, collagen is a very good carrier for bonemorphogenic proteins, when bone morphogenic proteins are added tocollagen sponges, the original bonding found in nature is not recreatedand, as a result the bone morphogenic proteins are only physicallyincorporated in the collagen sponges and become released too quickly,thereby failing to retain their full efficacy.

Sometimes when the surgeon manipulates the matrix to place it in thebone defect, excessive amounts of growth factor (e.g., bone morphogenicprotein) may leak or migrate from the matrix, which may reduce a stablemicroenvironment for new bone and/or cartilage growth. This migration ofthe bioactive agent may also cause the collagen sponge to fail to retainits full efficacy over time to maximally promote bone and/or cartilagegrowth at a target site, side effects can be exaggerated and dosagesrequired for treatment become uneconomically large.

Thus, there is a need to develop new osteogenic compositions and methodsthat improve bone and/or cartilage repair by retaining bone morphogenicproteins and other growth factors much longer than prior art collagensponges. It is, therefore, desirable to provide methods of preparingbone material having increased surface area, increased biologicalactivities including but not limited to osteoinductive activity.Further, it is also desirable to provide bone implants prepared frombone material having enhanced osteoinductivity and enhanced ability togrow and integrate into a host bone.

SUMMARY

Methods for making a biodegradable collagen matrix having increasedosteoinductivity are provided. In various embodiments, the methodsinclude providing an acidic collagen slurry which is mixed with at leastone water soluble and/or hydrophilic bioactive agent under conditionssufficient to cause the collagen slurry to self-assemble intomacroscopic collagen fibers and cause the at least one bioactive agentto form a collagen matrix containing the bioactive agent. Conditionssufficient to cause the collagen slurry to self-assembly include raisingthe pH of the slurry to from about 5 to about a pH of 9; and/or addingbone powder, calcium phosphate, hydroxyapatite, DBM or a mixture thereofto the acidic collagen slurry in order to raise the pH from about 5 toabout 9.

In other embodiments, the method for making a biodegradable collagenmatrix having increase osteoinductivity includes adding a dried mixtureof a bioactive agent and bone powder, calcium phosphate, hydroxyapatite,DBM or a mixture thereof to an acidic collagen slurry to form a collagenmatrix of macroscopic collagen fibers wherein the bioactive agent isbound to the macroscopic collagen fibers. In some embodiments, themethod for making a biodegradable collagen matrix further includesremoving water from the collagen matrix containing the bioactive agent.In yet other embodiments, the method for making a biodegradable collagenmatrix further comprises adding glycerol to the dried collagen matrix toform a malleable complex that does not harden. In various otherembodiments, the dried mixture of a bioactive agent and bone powder,calcium phosphate, hydroxyapatite, DBM or a mixture thereof is added tothe acidic collagen slurry prior to use at a surgical site by a medicalprofessional.

The present application also provides a biodegradable collagen matrixfor delivery of a bioactive agent. The collagen matrix includes aplurality of macroscopic collagen fibers comprising the bioactive agentbound to the macroscopic collagen fibers and is precipitated from anacidic collagen slurry by causing the pH of the slurry to be raisedabove a pH from about 5 to about a pH of 9.

In certain embodiments, the bioactive agent bound onto the macroscopicfibers of the collagen matrix comprise a growth factor, a bonemorphogenetic protein, an analgesic, an anti-inflammatory, andantibiotic, a cytokine, a chemotherapeutic or a mixture thereof In someembodiments, the bone morphogenetic protein bound to the macroscopicfibers of the collagen matrix can be BMP-2.

In other embodiments, a biodegradable collagen matrix for delivery of abioactive agent is provided, wherein the collagen matrix comprises aplurality of macroscopic collagen fibers having the bioactive agentbound to the macroscopic collagen fibers. In these embodiments, thecollagen matrix has a structure that results from reacting an acidiccollagen slurry or suspension with at least one water soluble and/orhydrophilic bioactive agent so as to raise the pH of the slurry above 5to about 9 sufficient to cause the collagen slurry to self-assemble intoa collagen matrix of macroscopic collagen fibers.

In other embodiments, the methods described herein contemplate furtheradding an osteoinductive additive comprising bone marrow aspirant,blood, blood products, synthetic and naturally-derived bone morphogenicproteins, growth factors, particulate demineralized bone matrix, ormixtures thereof In other embodiments, the methods described hereincontemplate further adding an osteoconductive additive, theosteoconductive additive comprising calcium phosphates, calcium sulfate,particulate demineralized bone matrix, naturally-derived allogenic bonemineral, naturally-derived autogenic bone mineral or mixtures thereof.

In one embodiment, there is a biodegradable collagen matrix for deliveryof a bioactive agent, the collagen matrix comprising a plurality ofmacroscopic collagen fibers comprising the bioactive agent bound to themacroscopic collagen fibers, wherein the collagen matrix has a structurethat results from reacting an acidic collagen slurry (e.g., collagensuspension) with at least one water soluble and/or hydrophilic bioactiveagent so as to raise the pH of the slurry above 5 to about 9 sufficientto cause the collagen slurry to self-assemble into a collagen matrix ofmacroscopic collagen fibers.

While multiple embodiments are disclosed, still other embodiments of thepresent disclosure will become apparent to those skilled in the art fromthe following detailed description, which shows and describesillustrative embodiments of the disclosure. As will be realized, thevarious embodiments of the present disclosure are capable ofmodifications in various obvious aspects, all without departing from thespirit and scope of the present disclosure. Accordingly, the detaileddescription is to be regarded as illustrative in nature and notrestrictive.

DETAILED DESCRIPTION Definitions

For the purposes of this specification and appended claims, unlessotherwise indicated, all numbers expressing quantities of ingredients,percentages or proportions of materials, reaction conditions, and othernumerical values used in the specification and claims, are to beunderstood as being modified in all instances by the term “about.”Similarly, when values are expressed as approximations, by use of theantecedent “about,” it will be understood that the particular valueforms another embodiment that is +/−10% of the recited value.Accordingly, unless indicated to the contrary, the numerical parametersset forth in the following specification and attached claims areapproximations that may vary depending upon the desired propertiessought to be obtained by the present disclosure. At the very least, andnot as an attempt to limit the application of the doctrine ofequivalents to the scope of the claims, each numerical parameter shouldat least be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Also, as used inthe specification and including the appended claims, the singular forms“a,” “an,” and “the” include the plural, and reference to a particularnumerical value includes at least that particular value, unless thecontext clearly dictates otherwise. Ranges may be expressed herein asfrom “about” or “approximately” one particular value and/or to “about”or “approximately” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of this application are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements. Moreover, all ranges disclosed hereinare to be understood to encompass any and all subranges subsumedtherein. For example, a range of “1 to 10” includes any and allsubranges between (and including) the minimum value of 1 and the maximumvalue of 10, that is, any and all subranges having a minimum value ofequal to or greater than 1 and a maximum value of equal to or less than10, e.g., 5.5 to 10.

“Bioactive agent” or “bioactive compound” is used herein to refer to acompound or entity that alters, inhibits, activates, or otherwiseaffects biological or chemical events. For example, bioactive agents mayinclude, but are not limited to, osteogenic or chondrogenic proteins orpeptides, anti-AIDS substances, anti-cancer substances, antibiotics,immunosuppressants, anti-viral substances, enzyme inhibitors, hormones,neurotoxins, opioids, hypnotics, anti-histamines, lubricants,tranquilizers, anti-convulsants, muscle relaxants and anti-Parkinsonsubstances, anti-spasmodics and muscle contractants including channelblockers, miotics and anti-cholinergics, anti-glaucoma compounds,anti-parasite and/or anti-protozoal compounds, modulators ofcell-extracellular matrix interactions including cell growth inhibitorsand antiadhesion molecules, vasodilating agents, inhibitors of DNA, RNAor protein synthesis, anti-hypertensives, analgesics, anti-pyretics,steroidal and non-steroidal anti-inflammatory agents, anti-angiogenicfactors, angiogenic factors, anti-secretory factors, anticoagulantsand/or antithrombotic agents, local anesthetics, ophthalmics,prostaglandins, anti-depressants, anti-psychotic substances,anti-emetics, and imaging agents. In certain embodiments, the bioactiveagent is a drug. In certain embodiments, the bioactive agent is a smallmolecule. Bioactive agents further include RNAs, such as siRNA, andosteoclast stimulating factors. In some embodiments, the bioactive agentmay be a factor that stops, removes, or reduces the activity of bonegrowth inhibitors. In some embodiments, the bioactive agent is a growthfactor, cytokine, extracellular matrix molecule or a fragment orderivative thereof, for example, a cell attachment sequence such as RGD.

A more complete listing of bioactive agents and specific drugs suitablefor use in the present application may be found in “PharmaceuticalSubstances: Syntheses, Patents, Applications” by Axel Kleemann andJurgen Engel, Thieme Medical Publishing, 1999; the “Merck Index: AnEncyclopedia of Chemicals, Drugs, and Biologicals”, edited by SusanBudavari et al., CRC Press, 2013; and the United StatesPharmacopeia-37/National Formulary-32, published by the United StatesPharmacopeia Convention, Inc., Rockville Md., 2014, all of which areincorporated herein by reference. Drugs for human use listed by the U.S.Food and Drug Administration (FDA) under 21 C.F.R. §§330.5, 331 through361, and 440 hrough 460, and drugs for veterinary use listed by the FDAunder 21 C.F.R. §§500 through 589, all of which are incorporated hereinby reference, are also considered acceptable for use in accordance withthe present invention.

“Biodegradable”, “bioerodable”, or “resorbable” materials are materialsthat degrade under physiological conditions to form a product that canbe metabolized or excreted without damage to the subject. In certainembodiments, the product is metabolized or excreted without permanentdamage to the subject. Biodegradable materials may be hydrolyticallydegradable, may require cellular and/or enzymatic action to fullydegrade, or both. Biodegradable materials also include materials thatare broken down within cells. Degradation may occur by hydrolysis,enzymatic processes, phagocytosis, or other processes.

“Biocompatible,” as used herein, is intended to describe materials that,upon administration in vivo, do not induce undesirable long-termeffects.

“Bone,” as used herein, refers to bone that is cortical, cancellous orcortico-cancellous of autogenous, allogenic, xenogenic, or transgenicorigin.

“Demineralized,” as used herein, refers to any material generated byremoving mineral material from tissue, for example, bone tissue. Incertain embodiments, the demineralized compositions described hereininclude preparations containing less than 5% calcium. “Demineralized” isintended to encompass such expressions as “substantially demineralized,”“partially demineralized,” “surface demineralized,” and “fullydemineralized.” “Partially demineralized” is intended to encompass“surface demineralized.”

“Demineralized bone activity” refers to the osteoinductive activity ofdemineralized bone.

“Demineralized bone matrix (DBM),” as used herein, refers to anymaterial generated by removing mineral material from bone tissue. Insome embodiments, the DBM compositions as used herein includepreparations containing less than 5% calcium and, in some embodiments,less than 1% calcium by weight. In other embodiments, the DBMcompositions comprise partially demineralized bone (e.g., preparationswith greater than 5% calcium by weight but containing less than 100% ofthe original starting amount of calcium).

The term “lyophilized” or “freeze-dried” includes a state of a substancethat has been subjected to a drying procedure such as lyophilization,where at least 50% of moisture has been removed. The growth factor maybe lyophilized or freeze-dried.

“Mammal,” as used herein refers to organisms from the taxonomy class“mammalian,” including but not limited to humans, other primates such aschimpanzees, apes, orangutans and monkeys, rats, mice, cats, dogs, cows,or horses.

“Osteoconductive,” as used herein, refers to the ability of a substanceto serve as a template or substance along which bone may grow.

“Osteogenic,” as used herein, refers to materials containing livingcells capable of differentiation into bone tissue.

“Osteoimplant,” as used herein, refers to any implant prepared inaccordance with the embodiments described herein and therefore mayinclude expressions such as bone material, bone membrane, bone graft.

“Osteoinductive,” as used herein, refers to the quality of being able torecruit cells from the host that have the potential to stimulate newbone formation. Any material that can induce the formation of ectopicbone in the soft tissue of an animal is considered osteoinductive. Forexample, most osteoinductive materials induce bone formation in athymicrats when assayed according to the method of Edwards et al.,“Osteoinduction of Human Demineralized Bone: Characterization in a RatModel,” Clinical Orthopaedics & Rel. Res., 357:219-228, December 1998,incorporated herein by reference.

In other instances, osteoinduction is considered to occur throughcellular recruitment and induction of the recruited cells to anosteogenic phenotype. Osteoinductivity score refers to a score rangingfrom 0 to 4 as determined according to the method of Edwards et al.(1998) or an equivalent calibrated test. In the method of Edwards etal., a score of “0” represents no new bone formation; “1” represents1%-25% of implant involved in new bone formation; “2” represents 26-50%of implant involved in new bone formation; “3” represents 51%-75% ofimplant involved in new bone formation; and “4” represents >75% ofimplant involved in new bone formation. In most instances, the score isassessed 28 days after implantation. However, the osteoinductivity scoremay be obtained at earlier time points such as 7, 14, or 21 daysfollowing implantation. In these instances it may be desirable toinclude a normal DBM control such as DBM powder without a carrier, andif possible, a positive control such as BMP. Occasionallyosteoinductivity may also be scored at later time points such as 40, 60,or even 100 days following implantation. Percentage of osteoinductivityrefers to an osteoinductivity score at a given time point expressed as apercentage of activity, of a specified reference score. Osteoinductivitymay be assessed in an athymic rat or in a human. Generally, as discussedherein, an osteoinductive score is assessed based on osteoinductivity inan athymic rat.

“Superficially demineralized,” as used herein, refers to bone-derivedelements possessing at least about 90 weight percent of their originalinorganic mineral content, the expression “partially demineralized” asused herein refers to bone-derived elements possessing from about 8 toabout 90 weight percent of their original inorganic mineral content andthe expression “fully demineralized” as used herein refers to bonecontaining less than 8% of its original mineral context.

A “therapeutically effective amount” or “effective amount” is such thatwhen administered, the drug results in alteration of the biologicalactivity, such as, for example, promotion of bone, cartilage and/orother tissue (e.g., vascular tissue) growth, inhibition of inflammation,reduction or alleviation of pain, improvement in the condition throughinhibition of an immunologic response. The dosage administered to apatient can be as single or multiple doses depending upon a variety offactors, including the drug's administered pharmacokinetic properties,the route of administration, patient conditions and characteristics(sex, age, body weight, health, size, etc.), extent of symptoms,concurrent treatments, frequency of treatment and the effect desired.

The terms “treating” and “treatment” when used in connection with adisease or condition refer to executing a protocol that may includeosteochondral repair procedure, administering one or more drugs to apatient (human or other mammal), in an effort to alleviate signs orsymptoms of the disease or condition or immunological response.Alleviation can occur prior to signs or symptoms of the disease orcondition appearing, as well as after their appearance. Thus, treatingor treatment includes preventing or prevention of disease or undesirablecondition. In addition, treating, treatment, preventing or prevention donot require complete alleviation of signs or symptoms, does not requirea cure, and specifically includes protocols that have only a marginaleffect on the patient. In some embodiments, the osteogenic compositioncan be used to treat subchondral, osteochondral, hyaline cartilageand/or condyle defects.

Collagen Matrix and Methods of Preparation

In various aspects, a biodegradable collagen matrix for delivery of abioactive agent is provided. The collagen matrix comprises a pluralityof macroscopic collagen fibers having the bioactive agent bound to themacroscopic collagen fibers, the collagen matrix precipitated from anacidic collagen slurry by causing the pH of the slurry to be raisedabove a pH from about 5 to about a pH of 9.

In other embodiments, a biodegradable collagen matrix for delivery of abioactive agent is provided wherein the collagen matrix comprises aplurality of macroscopic collagen fibers comprising the bioactive agentbound to the macroscopic collagen fibers. In these embodiments, thecollagen matrix has a structure that results from reacting an acidiccollagen slurry or suspension with at least one water soluble and/orhydrophilic bioactive agent so as to raise the pH of the slurry above 5to about 9, which is sufficient to cause the collagen slurry toself-assemble into a collagen matrix of macroscopic collagen fibers.

In one embodiment, the present application provides a method for makinga biodegradable collagen matrix. The method includes providing an acidiccollagen slurry or suspension, and mixing the slurry or suspension withat least one water soluble or hydrophilic bioactive agent underconditions sufficient to cause the collagen slurry to self-assemble intomacroscopic collagen fibers and cause the at least one bioactive agentto form a collagen matrix wherein the bioactive agent becomes uniformlydistributed. Conditions sufficient to cause the collagen slurry toself-assembly comprise raising the pH of the slurry to from about 5 toabout a pH of 9 and/or adding bone powder, calcium phosphate,hydroxyapatite, DBM or a mixture thereof to the slurry to raise the pHfrom about 5 to about 9.

In some embodiments, collagen suspensions or slurries are made bybreaking down collagen bearing tissues into small pieces and mixing theminto an acid solution which results in a stable suspension or slurry. Insome embodiments, in an acidic environment, as the pH is raised,collagen molecules react and bond with each other by displacing water toform collagen precipitates which self-assemble into a collagen matrix inthe absence of a cross-linking agent.

In other embodiments, in an acidic environment, the collagen moleculescan also react with other soluble or hydrophilic materials havingnegatively charged surface groups such as compounds containinghydroxides or amines. Materials capable of forming negatively chargedsurface groups such as hydroxides or amines comprise without limitationsbioactive agents or bone particles, demineralized bone particles,calcium phosphates that can then react with the collagen molecules tobind and/or adhere to the macroscopic fibers of the collagen matrix. Insome embodiments, the binding between the bioactive agent (e.g., watersoluble and /or hydrophilic bioactive agent, such as for example, agrowth factor, BMP, analgesic, anti-inflammatory agent, antibiotic,cytokine, chemotherapeutic agent, etc.) can be electrostaticinteractions, hydrogen bonding, van der Waals interaction, hydrophobicinteraction, hydrophilic interaction, covalent bonding, or non-covalentbonding.

Hydrophilic growth factors are protein based bioactive agents which arecapable of binding to the collagen suspensions. In various embodiments,the growth factor(s) can be added to the acidic collagen slurry beforethe slurry or suspension is neutralized and precipitated. The growthfactor(s) will then be incorporated chemically into the resultingcollagen matrix, or into a collagen matrix that includes anothersubstance such as bone, DBM, or calcium phosphates. The resultingcollagen matrix will release the growth factor more slowly than if itwere added to a fully formed collagen sponge, membrane, or composite.

Upon neutralization, namely as the pH is raised above 5 to about 9, thecollagen slurry is destabilized and the collagen spontaneously assemblesinto macroscopic fibers. The precipitation of collagen or collagencomplexes is accompanied by the release of free water because the waterbonding sites on the collagen are now binding to chemically similar towater sites on the complexing material. Further evidence of a chemicalinteraction in the collagen precipitation is provided by the observationthat the precipitated collagen complexes, when physically pulled apart,cannot recombine to form a material with the original degree ofcohesiveness.

In some embodiments, to ensure greater storage stability, the waterformed during the formation of the collagen matrix can be removed, andthe collagen matrix can be dried prior to use, for example bylyophilization. In other embodiments, if a malleable collagen matrix isdesired which does not harden, then before use without the need forrehydration, a suitable liquid such as glycerol can be added to the wetbioactive agent —collagen complex before lyophilization. Lyophilizationwill then remove the water but leave the added glycerol liquid. Theadded glycerol should be chosen based on its ability to preserve, or atleast not degrade, the bioactive agent.

In other embodiments, the collagen matrix of this application can beprepared by other methods. In one aspect, the biodegradable collagenmatrix is prepared by adding a dried mixture of a bioactive agent andbone powder, calcium phosphate, hydroxyapatite, DBM or a mixture thereofto an acidic collagen slurry to form a collagen matrix of macroscopiccollagen fibers wherein the bioactive agent is bound to the macroscopiccollagen fibers and uniformly distributed therein. In this aspect, theresulting collagen matrix will release the bioactive agent more slowlythan if it were added to a fully formed collagen sponge, membrane orcomposite. Therefore, the bioactive agent is reacted with the formingcollagen matrix structure and the structure of the matrix results fromits reaction with the bioactive agent during collagen matrix formation.

As with other methods described herein, to ensure greater storagestability, the water formed during the formation of the collagen matrixcan be removed, and the collagen matrix can be dried prior to use, forexample by lyophilization. In other embodiments, if a malleable collagenmatrix is desired which does not harden, then before use and without theneed for rehydration, a suitable liquid such as glycerol can be added tothe wet bioactive agent—collagen complex before lyophilization.Lyophilization will then remove the water but leave the added glycerolliquid. In some embodiments, the dried mixture of bioactive agent andbone powder, calcium phosphate, hydroxyapatite, DBM or a mixture thereofcan be added to the acidic collagen slurry at the point of use, orearlier, if desired by the appropriate medical professional.

In various aspects, the biodegradable collagen matrix obtained by themethods of this application comprises, consists essentially of, orconsists of macroscopically visible collagen fiber, namely, collagenfibers visible with the naked eye to which is bonded a bioactive agentuniformly distributed therein. The mean length of the collagen fibers isgenerally from about 3 to about 30 mm, in some embodiments, from about 5to about 25 mm, and, in other embodiments, from about 7.5 to about 20mm.

There are many advantages to the collagen matrices containing thebioactive agent prepared by the methods described herein. It has beenunexpectedly found, that these collagen matrices mimic the naturaldelivery of many bioactive agents and especially growth factors to abone defect site. Moreover, these collagen matrices can act as ascaffold for tissue growth and are fully resorbable and/or remodelableand exhibit excellent biocompatibility with all tissues.

Collagen

The collagen in the collagen matrix of this application can be derivedfrom any collagen bearing tissue from an animal. The collagen can beallogenic or xenogenic. The collagen can be from skin, tendon, fascia,ligament, trachea, organ collagen, etc. In certain embodiments, thecollagen is human collagen or other mammalian collagen (e.g., porcine,bovine, or ovine). The collagen can be sourced from any animal.

If human sourced collagen is used, some of the common processing steps,designed to remove immunogenic proteins, can optionally be skipped.Steps to remove immune proteins typically include treatment with aprotolytic enzyme such as papen, or ficin, treatment with a strongoxidizing agent such as sodium chlorate, and exposure to high pH from,for example, sodium hydroxide. If any of these protein destroying stepsare to be carried out, they must be completed, and all residual reagentsbe removed, before the growth factor(s) is added to the forming collagenmatrix.

Presently, about twenty eight distinct collagen types have beenidentified in vertebrates, including bovine, ovine, porcine, chicken,marine, and human sources. Generally, the collagen types are numbered byRoman numerals, and the chains found in each collagen type areidentified by Arabic numerals. Detailed descriptions of structure andbiological functions of the various different types of naturallyoccurring collagens are generally available in the art.

The collagen may have the same composition as in naturally occurringsources. Examples of sources of collagens include human or non-human(bovine, ovine, and/or porcine), as well as recombinant collagen orcombinations thereof. Examples of suitable collagen include, but are notlimited to, human collagen type I, human collagen type II, humancollagen type III, human collagen type IV, human collagen type V, humancollagen type VI, human collagen type VII, human collagen type VIII,human collagen type IX, human collagen type X, human collagen type XI,human collagen type XII, human collagen type XIII, human collagen typeXIV, human collagen type XV, human collagen type XVI, human collagentype XVII, human collagen type XVIII, human collagen type XIX, humancollagen type XXI, human collagen type XXII, human collagen type XXIII,human collagen type XXIV, human collagen type XXV, human collagen typeXXVI, human collagen type XXVII, and human collagen type XXVIII, orcombinations thereof. Collagen may further or alternatively comprisehetero- and homo-trimers of any of the above-recited collagen types. Insome embodiments, the collagen comprises hetero- or homo-trimers ofhuman collagen type I, human collagen type II, human collagen type III,or combinations thereof.

In some embodiments, the collagen is all type I or substantially all iscollagen type I, namely, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95%. Insome embodiments all soft tissue growth factors are conserved. In otherembodiments, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80% at least 85%, at least 90%, atleast 95%, or at least 99% of the tissue growth factors are conserved.

The collagen may be from any collagen containing organ source such asskin, fascia, intestine, tendon, bladder and trachea. In someembodiments, human compatible collagen, and xenograft collagen can beused if they can be rendered non-immunogenic by a process that does notdestroy the activity of the natural growth factors contained in thetissue or diminishes the activity by an acceptably small amount.Examples of processes that conserve natural growth factors include butare not limited to glycosidase digestion of carbohydrate moieties of thexenograft, which are optionally followed by treatment of carbohydratemoieties of the xenografts with a capping agent. Thus, sugars and othersubstances may be removed during processing, while the soft tissuegrowth factors remain associated with the collagen. In some embodimentssoft tissue growth factors are conserved, but any sugars have beenremoved or at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80% at least 85%, at least 90%, atleast 95%, or at least 99% of them have been removed.

Self-Assembly of Collagen

The collagen can be made into a water-based dispersion by an acidtreatment using techniques known to the art. As an example, a dispersioncan include about 0.2 mL of lactic acid in 100 mL ofdistilled/de-ionized water with about 1.0 g of collagen (e.g., skin,tendon, fascia, ligament). In some embodiments, an acidic dispersioncontains from about 0.1 wt % to about 3 wt % collagen, for example, fromabout 1 wt % (1 g collagen/100 mL solution) and 2 wt % collagen. Thecollagen particles (e.g., macroscopic fibers, strands) in the dispersionare capable of self-assembly in the absence of a cross-linking agent.

Collagen has the ability to self-assemble into fibers, and thisself-assembly has a significant role in maintaining the structure andthe mechanical function of a given tissue. Collagen self-assembly occursat a physiological pH where the assembled collagen fibers haveidentifiable quarter staggered configurations with alternating light anddark bands when observed under high magnifications. The regions wherethe collagen molecules overlap are the dark banding regions, while thestaggered collagen molecules spanning from dark band to dark band arethe light banding regions. Within the overlapping region, collagenmolecules are bound together by native cross-links that are formed aspart of fiber formation and overall molecule stabilization. Nativecross-links alone are not the only element that is maintaining thestability of collagen molecules. Hydrogen bonding between thesemolecules can be facilitated by water that can also play a role incollagen self-assembly. Water may also continue to influence collagenstability after self-assembly on the micro- and macro-molecular levelwhere water helps to maintain collagen molecular conformation andmechanical properties.

At a lower pH, collagen can disassemble into smaller fibril subunits.This physical transformation is termed swelling in acidic solutions.Without being bound by any particular theory, it is believed that themechanism is made possible by the charges on the collagen proteinbecoming positive, which causes the collagen fibers to repel each otherand deaggregate into subunits because the attractive forces of theinteraction between triple helices are eliminated, which can result in adispersion of insoluble collagen that is homogenous, opalescent, andoptically isotropic.

In some embodiments, the dispersed collagen includes long fibers withdiameters in the range of from about 0.05 μm to about 2.5 μm, andlengths from about 5 μm to about 100 μm. In various embodiments, thecollagen fibers are macroscopic, that is they can be seen with the nakedeye and their mean length of the collagen fibers is generally from about3 to about 30 mm, in some embodiments, from about 5 to about 25 mm, and,in other embodiments, from about 7.5 to about 20 mm.

The process of deaggregation is reversible when the collagen dispersionis brought to physiological pH with evident collagen fiber assembly,e.g., if the acidity of the dispersion has been maintained above pH 3.As a result, in some embodiments, the pH of the collagen dispersionranges from about 3 to about 6.5, for example, from about 3 to about 5,from about 3 to about 4.6, or about 4. As an example, about 0.2 mL oflactic acid in about 100 mL of water provides a pH from about 4 to about5.

The methods of preparing the osteoimplant compositions involve usingdifferent collagen characteristics under different pH conditions to formthe compositions. For example, bone particles, DBM or calcium phophatesthat are added to the acidic collagen dispersion slowly neutralize thedispersion as surface minerals from these sources are dissolved. Thegradual rise in pH allows collagen assembly to take place around thebone particles, which may have exposed collagen fibers on their surfacesas a result of the mineral dissolution.

In various embodiments, the dispersion is very pure and collagen fiberscan slowly be precipitated by drop-by-drop addition of an alkali, suchas for example, sodium hydroxide, sodium carbonate, ammonia, sodiumsulfate, or the like. Typical pH for this precipitation step is about 7.The collagen fibers can be filtered or collected by hand or machine.

In various embodiments for the collagen precipitation, the pH of theacidic dispersion of collagen is pH of about 3.5 as a starting point forthe precipitation reaction, at about pH of 4.6 transparent shard-likestructures form, these structures precipitate out of the acid dispersionat a pH of about 6.0 to about 7.0, where they are transparent fullyformed, firm and stable structures. In various embodiments, thetemperature for precipitation is about 20 to about 30° C.

In various embodiments, the collagen formed is transparent shard-likestructures resembling flexible icicles. The collagen fibers appear likeshard-like gelatin because it is thought that water is trapped withinthe fiber structure. The collagen fibers can be de-watered making thefiber structure more textile like and allows further removal ofcontaminants, such as non-collagenous material, trapped within thewater.

In various embodiments, dewatering of the collagen fibers can beaccomplished by, for example, centrifugation, washing with suitabledrying agents, air, and/or oven drying. Suitable drying agents include,for example, non-polar solvents such as for example, acetone, alcohol,or the like. Low temperature drying, such as by air and/or oven attemperatures, for example, of about 35 to about 40° C. can remove anyremaining water as the solvents flash off, and can leave substantiallypure collagen in dry firm fiber form.

Growth Factors

The growth factors include osteoinductive agents (e.g., agents thatcause new bone growth in an area where there was none) and/orosteoconductive agents (e.g., agents that cause ingrowth of cells) andalso fibrous or soft tissue inducing agents. Osteoinductive agents canbe polypeptides or polynucleotide compositions. Polynucleotidecompositions of the osteoinductive agents include, but are not limitedto, isolated Bone Morphogenic Protein (BMP), Vascular Endothelial GrowthFactor (VEGF), Connective Tissue Growth Factor (CTGF, which may bespecific for tendons and ligaments), Osteoprotegerin, GrowthDifferentiation Factors (GDFs), Cartilage Derived Morphogenic Proteins(CDMPs, which can be a foundation for soft or hard tissue), LimMineralization Proteins (LMPs), Platelet derived growth factor, (PDGF orrhPDGF, which is particularly advantageous for use with soft tissue),Insulin-like growth factor (IGF) or Transforming Growth Factor beta(TGF-beta) polynucleotides.

The collagen matrix may also comprise one or more additional growthfactors, including but not limited to BMP-2, rhBMP12 or BMP7. Theseadditional growth factors, unlike the conserved proteins are ones thathave been added to the collagen during the formation of the collagenmatrix and the matrix has these bioactive agents as part of itsstructure. The identity of proteins may be the same as or different thanthe conserved proteins. In some embodiments the collagen may be treatedso that it also binds to these additional proteins. In some embodiments,the resulting concentration of growth factors is from 10% to 30% greaterthan in the natural state or from 30% to 50% greater than in the naturalstate or from 50% to 70% greater than in the natural state.

Additional growth factors can include polynucleotide compositions.Polynucleotide compositions include, but are not limited to, genetherapy vectors harboring polynucleotides encoding the osteoinductivepolypeptide of interest. Gene therapy methods often utilize apolynucleotide that codes for the osteoinductive polypeptide operativelylinked to or associated with a promoter or any other genetic elementsnecessary for the expression of the osteoinductive polypeptide by thetarget tissue. Such gene therapy and delivery techniques are known inthe art (see, for example, International Publication No. WO90/11092, thedisclosure of which is herein incorporated by reference in itsentirety). Suitable gene therapy vectors include, but are not limitedto, gene therapy vectors that do not integrate into the host genome.Alternatively, suitable gene therapy vectors include, but are notlimited to, gene therapy vectors that integrate into the host genome.

In some embodiments, the polynucleotide can be delivered in plasmidformulations. Plasmid DNA or RNA formulations refer to polynucleotidesequences encoding osteoinductive polypeptides that are free from anydelivery vehicle that acts to assist, to promote or to facilitate entryinto the cell, including viral sequences, viral particles, liposomeformulations, lipofectin, precipitating agents or the like. Optionally,gene therapy compositions can be delivered in liposome formulations andlipofectin formulations, which can be prepared by methods well known tothose skilled in the art. General methods are described, for example, inU.S. Pat. Nos. 5,593,972, 5,589,466, and 5,580,859, the disclosures ofwhich are herein incorporated by reference in their entireties. Genetherapy vectors further comprise suitable adenoviral vectors including,but not limited to for example, those described in U.S. Pat. No.5,652,224, which is herein incorporated by reference.

Additional growth factors also include but are not limited to isolatedpolynucleotides that encode Bone Morphogenic Protein (BMP), VascularEndothelial Growth Factor (VEGF), Connective Tissue Growth Factor(CTGF), Osteoprotegerin, Growth Differentiation Factors (GDFs),Cartilage Derived Morphogenic Proteins (CDMPs), Lim MineralizationProteins (LMPs), Platelet derived growth factor, (PDGF or rhPDGF),Insulin-like growth factor (IGF) or Transforming Growth Factor beta(TGF-beta707) polypeptides. Polypeptide compositions of theosteoinductive agents also include, but are not limited to, full lengthproteins, fragments or variants thereof.

Variants of the isolated osteoinductive agents include, but are notlimited to, polypeptide variants that are designed to increase theduration of activity of the osteoinductive agent in vivo. Typically,variant osteoinductive agents include, but are not limited to, fulllength proteins or fragments thereof that are conjugated to polyethyleneglycol (PEG) moieties to increase their half-life in vivo (also known aspegylation). Methods of pegylating polypeptides are well known in theart (See, e.g., U.S. Pat. No. 6,552,170 and European Pat. No. 0,401,384as examples of methods of generating pegylated polypeptides). In someembodiments, the isolated osteoinductive agent(s) are provided as fusionproteins. In one embodiment, the osteoinductive agent(s) are availableas fusion proteins with the Fc portion of human IgG. In anotherembodiment, the osteoinductive agent(s) are available as hetero- orhomodimers or multimers. Examples of some fusion proteins include, butare not limited to, ligand fusions between mature osteoinductivepolypeptides and the Fc portion of human Immunoglobulin G (IgG). Methodsof making fusion proteins and constructs encoding the same are wellknown in the art.

Isolated osteoinductive agents that may be included within the collagenmatrix are typically sterile. In a non-limiting method, sterility isreadily accomplished for example by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes or filters). In oneembodiment, the collagen matrix includes osteoinductive agentscomprising one or more members of the family of Bone MorphogenicProteins (“BMPs”). BMPs are a class of proteins thought to haveosteoinductive or growth-promoting activities on endogenous bone tissue,or function as pro-collagen precursors. Known members of the BMP familyinclude, but are not limited to, BMP-1, BMP-2, BMP-3, BMP-4, BMP-5,BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-15,BMP-16, BMP-17, BMP-18 as well as polynucleotides or polypeptidesthereof, as well as mature polypeptides or polynucleotides encoding thesame. In one embodiment, the at least one bone morphogenic protein isBMP-2.

BMPs utilized as osteoinductive agents may comprise one or more ofBMP-1; BMP-2; BMP-3; BMP-4; BMP-5; BMP-6; BMP-7; BMP-8; BMP-9; BMP-10;BMP-11; BMP-12; BMP-13; BMP-15; BMP-16; BMP-17; or BMP-18; as well asany combination of one or more of these BMPs, including full length BMPsor fragments thereof, or combinations thereof, either as polypeptides orpolynucleotides encoding the polypeptide fragments of all of the recitedBMPs. The isolated BMP osteoinductive agents may be administered aspolynucleotides, polypeptides, full length protein or combinationsthereof.

In another embodiment, isolated osteoinductive agents that are includedin the collagen matrix include osteoclastogenesis inhibitors to inhibitbone resorption of the bone tissue surrounding the site of implantationby osteoclasts. Osteoclast and osteoclastogenesis inhibitors include,but are not limited to, osteoprotegerin polynucleotides or polypeptides,as well as mature osteoprotegerin proteins, polypeptides orpolynucleotides encoding the same. Osteoprotegerin is a member of theTNF-receptor superfamily and is an osteoblast-secreted decoy receptorthat functions as a negative regulator of bone resorption. This proteinspecifically binds to its ligand, osteoprotegerin ligand (TNFSF11/OPGL),both of which are key extracellular regulators of osteoclastdevelopment.

Osteoclastogenesis inhibitors that can be loaded in the collagen matrixfurther include, but are not limited to, chemical compounds such asbisphosphonate, 5-lipoxygenase inhibitors such as those described inU.S. Pat. Nos. 5,534,524 and 6,455,541 (the contents of which are hereinincorporated by reference in their entireties), heterocyclic compoundssuch as those described in U.S. Pat. No. 5,658,935 (herein incorporatedby reference in its entirety), 2,4-dioxoimidazolidine and imidazolidinederivative compounds such as those described in U.S. Pat. Nos. 5,397,796and 5,554,594 (the contents of which are herein incorporated byreference in their entireties), sulfonamide derivatives such as thosedescribed in U.S. Pat. No. 6,313,119 (herein incorporated by referencein its entirety), or acylguanidine compounds such as those described inU.S. Pat. No. 6,492,356 (herein incorporated by reference in itsentirety).

In another embodiment, isolated osteoinductive agents that can be loadedin the collagen matrix include one or more members of the family ofConnective Tissue Growth Factors (“CTGFs”). CTGFs are a class ofproteins thought to have growth-promoting activities on connectivetissues. Known members of the CTGF family include, but are not limitedto, CTGF-1, CTGF-2, CTGF-4 polynucleotides or polypeptides thereof, aswell as mature proteins, polypeptides or polynucleotides encoding thesame.

In another embodiment, isolated osteoinductive agents that can be loadedin the collagen matrix include one or more members of the family ofVascular Endothelial Growth Factors (“VEGFs”). VEGFs are a class ofproteins thought to have growth-promoting activities on vasculartissues. Known members of the VEGF family include, but are not limitedto, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E or polynucleotides orpolypeptides thereof, as well as mature VEGF-A, proteins, polypeptidesor polynucleotides encoding the same.

In another embodiment, isolated osteoinductive agents that can be loadedin the collagen matrix include one or more members of the family ofTransforming Growth Factor-beta (“TGF-betas”). TGF-betas are a class ofproteins thought to have growth-promoting activities on a range oftissues, including connective tissues. Known members of the TGF-betafamily include, but are not limited to, TGF-beta-1, TGF-beta-2,TGF-beta-3, polynucleotides or polypeptides thereof, as well as matureprotein, polypeptides or polynucleotides encoding the same.

In another embodiment, isolated osteoinductive agents that can be loadedin the collagen matrix include one or more Growth DifferentiationFactors (“GDFs”). Known GDFs include, but are not limited to, GDF-1,GDF-2, GDF-3, GDF-7, GDF-10, GDF-11, and GDF-15. For example, GDFsuseful as isolated osteoinductive agents include, but are not limitedto, the following GDFs: GDF-1 polynucleotides or polypeptidescorresponding to GenBank Accession Numbers M62302, AAA58501, andAAB94786, as well as mature GDF-1 polypeptides or polynucleotidesencoding the same. GDF-2 polynucleotides or polypeptides correspondingto GenBank Accession Numbers BC069643, BC074921, Q9UK05, AAH69643, orAAH74921, as well as mature GDF-2 polypeptides or polynucleotidesencoding the same. GDF-3 polynucleotides or polypeptides correspondingto GenBank Accession Numbers AF263538, BCO30959, AAF91389, AAQ89234, orQ9NR23, as well as mature GDF-3 polypeptides or polynucleotides encodingthe same. GDF-7 polynucleotides or polypeptides corresponding to GenBankAccession Numbers AB158468, AF522369, AAP97720, or Q7Z4P5, as well asmature GDF-7 polypeptides or polynucleotides encoding the same. GDF-10polynucleotides or polypeptides corresponding to GenBank AccessionNumbers BCO28237 or AAH28237, as well as mature GDF-10 polypeptides orpolynucleotides encoding the same. GDF-11 polynucleotides orpolypeptides corresponding to GenBank Accession Numbers AF100907,NP_(—)005802 or 095390, as well as mature GDF-11 polypeptides orpolynucleotides encoding the same. GDF-15 polynucleotides orpolypeptides corresponding to GenBank Accession Numbers BC008962,BC000529, AAH00529, or NP_(—)004855, as well as mature GDF-15polypeptides or polynucleotides encoding the same.

In another embodiment, isolated osteoinductive agents that can be loadedin the collagen matrix include Cartilage Derived Morphogenic Protein(CDMP) and Lim Mineralization Protein (LMP) polynucleotides orpolypeptides. Known CDMPs and LMPs include, but are not limited to,CDMP-1, CDMP-2, LMP-1, LMP-2, or LMP-3.

CDMPs and LMPs useful as isolated osteoinductive agents that can beloaded in the collagen matrix include, but are not limited to, thefollowing CDMPs and LMPs: CDMP-1 polynucleotides and polypeptidescorresponding to GenBank Accession Numbers NM_(—)000557, U13660,NP_(—)000548 or P43026, as well as mature CDMP-1 polypeptides orpolynucleotides encoding the same. CDMP-2 polypeptides corresponding toGenBank Accession Numbers or P55106, as well as mature CDMP-2polypeptides. LMP-1 polynucleotides or polypeptides corresponding toGenBank Accession Numbers AF345904 or AAK30567, as well as mature LMP-1polypeptides or polynucleotides encoding the same. LMP-2 polynucleotidesor polypeptides corresponding to GenBank Accession Numbers AF345905 orAAK30568, as well as mature LMP-2 polypeptides or polynucleotidesencoding the same. LMP-3 polynucleotides or polypeptides correspondingto GenBank Accession Numbers AF345906 or AAK30569, as well as matureLMP-3 polypeptides or polynucleotides encoding the same.

In another embodiment, isolated osteoinductive agents that can be loadedin the matrix include one or more members of any one of the families ofBone Morphogenic Proteins (BMPs), Connective Tissue Growth Factors(CTGFs), Vascular Endothelial Growth Factors (VEGFs), Osteoprotegerin orany of the other osteoclastogenesis inhibitors, Growth DifferentiationFactors (GDFs), Cartilage Derived Morphogenic Proteins (CDMPs), LimMineralization Proteins (LMPs), or Transforming Growth Factor-betas(TGF-betas), as well as mixtures or combinations thereof.

In another embodiment, the one or more isolated osteoinductive agentsthat can be loaded in the collagen matrix are selected from the groupconsisting of BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8,BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-15, BMP-16, BMP-17, BMP-18,or any combination thereof; CTGF-1, CTGF-2, CGTF-3, CTGF-4, or anycombination thereof; VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, or anycombination thereof; GDF-1, GDF-2, GDF-3, GDF-7, GDF-10, GDF-11, GDF-15,or any combination thereof; CDMP-1, CDMP-2, LMP-1, LMP-2, LMP-3, and/orany combination thereof; Osteoprotegerin; TGF-beta-1, TGF-beta-2,TGF-beta-3, or any combination thereof; or any combination of one ormore members of these groups.

Other Additives

In various embodiments, the acidic collagen slurry can self-assembleupon addition of bone powder, calcium phosphate, hydroxyapatite, DBM ormixtures thereof. When added to the acidic collagen slurry, thesecompounds are all capable of raising the pH from about 5 to about 9,thereby causing the acidic collagen slurry to self-assemble intomacroscopic collagen fibers forming the collagen matrix describedherein. These bioactive agents are now part of the collagen matrixstructure.

The matrices of the current application do not have the bioactive agentloosely associated with them. Rather, the bioactive agent is bound inthe matrix and forms part of the matrix's structure.

In various embodiments, calcium phosphates can be used in forming thematrix. Calcium phosphate can be obtained from calcium phosphateceramics. Examples of such calcium phosphate ceramics include calciumphosphate compounds and salts, and combinations thereof, including:tricalcium phosphate Ca₃(PO₄)₂ (TCP), including alpha-TCP, beta-TCP, andbiphasic calcium phosphate containing alpha- and beta-TCP; amorphouscalcium phosphate (ACP); monocalcium phosphate Ca(H₂PO₄)₂ (MCP) andmonocalcium phosphate monohydrate Ca(H₂PO₄)₂H₂O (MCPM); dicalciumphosphate CaHPO₄ (DCP) and dicalcium phosphate dihydrate CaBPO42H₂O(DCPD); tetracalcium phosphate Ca₄(PO₄) ₂O(TTCP); octacalcium phosphateCa₈(PO₄)₄(HPO₄)₂.5H₂O (OCP); calcium hydroxyapatite Ca₁₀(PO₄)₆(OH)₂(CHA); calcium oxyapatite Ca₁₀(PO₄)₆O (COXA); calcium carbonate apatiteCa₁₀(PO₄)₆CO₃ (CCA); and calcium carbonate hydroxyapatites, e.g.,Ca₁₀(PO₄)₅(OH)(CO₃)₂ and Ca₁₀(PO₄)₄(OH)₂(CO₃)₃ (CCHA).

Calcium phosphates useful herein also include calcium-deficient calciumphosphates in which the molar or mass ratio of Ca:P is reduced by about20% or less, preferably about 15% or less, preferably about 10% or less,relative to the corresponding calcium non-deficient species, examples ofwhich include calcium-deficient hydroxyapatites, e.g.,Ca_(10-X)(HPO₄)_(X)(PO₄)_(6-X)(OH)_(2-X) (0≦X≦1) (CDHA);calcium-deficient carbonate hydroxyapatites (CDCHA); calcium-deficientcarbonate apatites (CDCA); and other calcium phosphate compounds andsalts known as useful in the bone graft material field, e.g., calciumpolyphosphates; and calcium-, phosphate-, and/or hydroxyl-“replaced”calcium phosphates, as further described below.

Calcium-replaced calcium phosphates are also useful herein, includinghomologs of any of the above in which some of, preferably a minority of(preferably about or less than: 40%, 35%, 33.3%, 30%, 25%, 20%, 15%, or10%) the calciums are substituted with monovalent and/or divalent metalcation(s), e.g., sodium calcium homologs thereof, such as CaNa(PO₄).

Phosphate-replaced calcium phosphates are also useful herein, includinghomologs of any of the above in which some of, preferably a minority of(preferably about or less than: 40%, 35%, 33.3%, 30%, 25%, 20%, 15%, or10%) the phosphate groups are substituted with carbonate, hydrogenphosphate, and/or silicate groups.

Demineralized bone matrix (DBM) can also be useful to raise the pH ofthe acidic collagen slurry while the collagen matrix is forming the DBMcan be used to raise the pH of the slurry from about 5 to about 9,thereby causing it to self-assemble into macroscopic collagen fibersforming the collagen matrix described herein.

DBM can be produced by acid extraction, thermal freezing, irradiation,or physical extraction of inorganic minerals from human or animal bone.The moisture level of the demineralized bone matrix can be easilycontrolled by air-drying or freeze-drying. Air dried demineralized bonematrix can include greater than about 10 weight percent of moisture,while in certain circumstances, freeze dried demineralized bone matrixcan include less than about 6 weight percent of moisture. In someaspects, DBM can include between about 5 and about 30 weight percent(e.g., between about 5-20 weight percent, between about 10-15 weightpercent, or between about 10-12 weight percent, or between about 5-10weight percent) of moisture, e.g., water. In various embodiments, thedemineralized bone matrix includes greater than or equal to about 6, 10,12, 14, 16, 18, 20, 22, 24, 26, or 28 weight percent of moisture; and/orless than or equal to about 30, 28, 26, 24, 22, 20, 18, 16, 14, 12, or 6weight percent of moisture. In some embodiments, the bone used tomanufacture the demineralized bone matrix can be cortical, cancellous,cortico-cancellous of autogenous, allogeneic, xenogeneic or transgenicin origin.

In various embodiments of the present teachings, demineralized bonematrix can be supplied as powdered cortical or cancellous bone or drychips ranging in size from about 10 μm to about 10 mm, from about 50 μmto about 5 mm, from about 100 μm to about 1 mm, from about 150 μm toabout 0.8 mm, or from about 200 μm to about 0.75 mm.

If desired, the collagen matrix of this application can be modified inother ways. In addition to growth factors, any useful biologicalsubstance that is water soluble or at least hydrophilic can be addedalone or in combination to the collagen as described above. The collagenshould bind to one or more the sites of hydration on the water solubleor hydrophilic material and so displace the water. If the material isnot naturally water soluble or hydrophilic, it can be derivatized orotherwise be made water soluble or hydrophilic before being combinedwith the collagen system. Types of useful water soluble/hydrophilicsubstances include, for example, antibiotics, pain relievers, growthfactors, anti-inflammatories, etc.

Medically/surgically useful substances which can be readily combinedwith the collagen during its manufacture include, for example, bonefibers, hydroxyapatite, and soluble solids and/or liquids dissolvedtherein, for example, antiviricides, particularly those effectiveagainst HIV and hepatitis; antimicrobials and/or antibiotics such aserythromycin, bacitracin, neomycin, penicillin, polymyxin B,tetracyclines, viomycin, chloromycetin and streptomycins, cefazolin,ampicillin, azactam, tobramycin, clindamycin and gentamycin; aminoacids, peptides, vitamins, inorganic elements, inorganic compounds,cofactors for protein synthesis, hormones; endocrine tissue or tissuefragments; synthesizers; enzymes such as collagenase, peptidases,oxidases; polymer cell scaffolds with paraenchymal cells; angiogenicdrugs and polymeric carriers containing such drugs; collagen lattices;biocompatible surface active agents; antigenic agents; cytoskeletalagents; cartilage fragments, living cells such as chondrocytes, bonemarrow cells, mesenchymal stem cells, natural extracts, tissuetransplants, bioadhesives, bone morphogenic proteins (BMPs),transforming growth factor (TGF-beta), insulin-like growth factor(IGF-1) (IGF-2), platelet derived growth factor (PDGF), fibroblastgrowth factors (FGF), vascular endothelial growth factor (VEGF),angiogenic agents, bone promoters, cytokines, interleukins, geneticmaterial, genes encoding bone promoting action, cells containing genesencoding bone promoting action; growth hormones such as somatotropin;bone digestors; antitumor agents; fibronectin; cellular attractants andattachment agents; immunosuppressants; permeation enhancers, forexample, fatty acid esters such as laureate, myristate and stearatemonesters of polyethylene glycol, surface active agents, enaminederivatives, α-keto aldehydes; nucleic acids; epidermal growth factor(EGF); all collagen types (not just type 1); non-collagenous proteinssuch as osteopontin, osteonectine, bone sialo proteins, vitronectin,thrombospondin, proteoglycans, decorin, biglycan, aggrecan, versican,tenascin, matrix gla protein hyaluronan; soluble and insolublecomponents of the immune system, soluble and insoluble receptorsincluding truncated forms, soluble, insoluble and cell surface boundligands including truncated forms; chemokines, bioactive compounds thatare endocytosed; compounds capable of altering the membrane potential ofcells, compounds capable of altering the monovalent and divalentcation/anion channels of cells; bone resorption inhibitors andstimulators; angiogenic and mitogenic factors; bioactive factors thatinhibit and stimulate second messenger molecules; integrin adhesionmolecules; clotting factors; externally expanded autograft or xenograftcells and any combinations thereof. The amounts of such optionally addedsubstances can vary widely with optimum levels being readily determinedin a specific case by routine experimentation.

Formation of an Implant

The collagen matrix containing a bioactive agent provided herein may beused to form an osteoinductive implant. The osteoimplant resulting fromthe mixture of the collagen matrix and/or bioactive agent, DBM,additive, may be flowable, have a putty consistency, may be shaped ormolded, and/or may be deformable but it will not harden. Theosteoimplant may assume a determined or regular form or configurationsuch as a sheet, plate, disk, tunnel, cone, or tube, to name but a few.Prefabricated geometry may include, but is not limited to, a crescentapron for single site use, an I-shape to be placed between teeth forintra-bony defects, a rectangular bib for defects involving both thebuccal and lingual alveolar ridges, neutralization plates,reconstructive plates, buttress plates, T-buttress plates, spoon plates,clover leaf plates, condylar plates, compression plates, bridge plates,or wave plates. Partial tubular as well as flat plates can be fabricatedfrom the osteoimplant. Such plates may include such conformations as,e.g., concave contoured, bowl shaped, or defect shaped. The osteoimplantcan be machined or shaped by any suitable mechanical shaping means.Computerized modeling can provide for the intricately-shapedthree-dimensional architecture of an osteoimplant custom-fitted to thebone repair site with great precision. In embodiments wherein theosteoimplant is shaped or moldable, the implant may retain coherence influids.

Accordingly, the osteoinductive collagen matrix may be subjected to aconfiguring step to form an osteoimplant. The configuring step can beemployed using conventional equipment known to those skilled in the artto produce a wide variety of geometries, e.g., concave or convexsurfaces, stepped surfaces, cylindrical dowels, wedges, blocks, screws,or the like. Also useful are demineralized bone and other matrixpreparations comprising additives or carriers such as binders, fillers,plasticizers, wetting agents, surface active agents, biostatic agents,biocidal agents, and the like. Some exemplary additives and carriersinclude polyhydroxy compounds, polysaccharides, glycosaminoglycanproteins, nucleic acids, polymers, polaxomers, resins, clays, calciumsalts, and/or derivatives thereof.

In some embodiments, the osteoinductive collagen matrix prepared from amixture of collagen slurry and bioactive agent and DBM may be placed ina containment device such as a porous mesh to provide a delivery system.In various embodiments, the device may comprise a polymer (such aspolyalkylenes (e.g., polyethylenes, polypropylenes, etc.), polyamides,polyesters, polyurethanes, poly(lactic acid-glycolic acid), poly(lacticacid), poly(glycolic acid), poly(glaxanone), poly(orthoesters),poly(pyrolicacid), poly(phosphazenes), L-co-G, etc.),other bioabsorbablepolymer such as Dacron or other known surgical plastics, a naturalbiologically derived material such as collagen, a ceramic (withbone-growth enhancers, hydroxyapatite, etc.), PEEK(polyether-etherketone), dessicated biodegradable material, metal,composite materials, a biocompatible textile (e.g., cotton, silk,linen), or other. In one embodiment, the containment device is formed asa long bag-like device and may be used with minimally invasivetechniques.

To facilitate on-site preparation and/or usage of the collagen matrixherein, the bioactive agent such as a growth factor, preferably inlyophilized or frozen form, and the collagen slurry, can be stored inseparate packages or containers under sterile conditions and broughttogether in intimate admixture at the moment of use for immediateapplication to an osseous defect site employing any suitable means suchas spatula, forceps, syringe, tamping device, or the like.

Alternatively, the collagen bioactive agent containing matrix can beprepared well in advance, lyophilized or otherwise dried and storedunder sterile conditions until required for use. In some embodiments,the collagen matrix described herein can be combined with autograft bonemarrow aspirate, autograft bone, preparations of selected autograftcells, autograft cells containing genes encoding bone promoting actionprior to being placed in a defect site. In various embodiments, theimplant composition is packaged already mixed and ready for use in asuitable container, such as for example, syringe, resealable non-toxicbottle, a bag mesh or pouch or is provided as a kit which can beprepared at a surgeon's direction when needed.

Having now generally described the invention, the same may be morereadily understood through the following reference to the followingexamples, which are provided by way of illustration and are not intendedto limit the present invention unless specified.

EXAMPLES Example 1

In this example, collagen matrix containing a bone morphogenic protein(BMP) is prepared according to a method described herein and its BMPcontent is compared to that present in a collagen matrix where the BMPis added after the formation of the collagen matrix, similar to adding aBMP to a collagen sponge wherein the BMP has not chemically combinedwith the collagen matrix.

A collagen suspension is made from human collagen having been minimallyprocessed and thus retains many immunogenic proteins. 1000 nanograms oflyophilized, water soluble BMP-2 is added to a 1% solution of suspendedcollagen (10 mg/I). The 1% collagen slurry is precipitated by combiningit with 100 milligrams of hydroxyapatite powder. Thecollagen-hydroxyapatite precipitate is squeezed dry, and then rinsedwith three 100 ml volumes of saline. After rinsing, thecollagen-hydroxyapatite collagen matrix is dried and digested. The BMP-2content of the collagen matrix is tested by ELISA assey.

By way of comparison, a control sample is also prepared. In the controlsample, the same collagen-hydroxyapatite precipitate is formed withoutBMP-2 present. After squeezing it dry, BMP-2 is added to precipitate,which was then washed with 100 ml of saline, dried, and tested for BMP-2by ELISA assey. The content of BMP-2 of the first sample is higher thanthat of the control sample.

Example 2

100 milligrams of hydroxyapatite is treated with 1000 nanograms of BMP-2dissolved in water. The hydroxyapatite with added BMP-2 is dried.Subsequently the hydroxyapatite BMP-2 dried mixture is added to acollagen slurry and a precipitate with collagen or collagen matrix isformed as described in Example 1. After rinsing with 100 ml of saline,the resulting collagen matrix is analyzed for BMP-2 content by ELISAassey. The BMP-2 content is similar to the content of BMP-2 obtainedfrom the first sample prepared in Example 1.

It will be understood that various modifications may be made to theembodiments disclosed herein. Therefore, the above description shouldnot be construed as limiting, but merely as exemplification of thevarious embodiments. Those skilled in the art will envision othermodifications within the scope and spirit of the claims appended hereto.

What is claimed is:
 1. A method for making a biodegradable collagenmatrix, the method comprising: providing an acidic collagen slurry; andmixing at least one water soluble and/or hydrophilic bioactive agentwith the acidic collagen slurry under conditions sufficient to cause thecollagen slurry to self-assemble into macroscopic collagen fibers andcause the at least one bioactive agent to form a collagen matrixcontaining the bioactive agent.
 2. A method for making a biodegradablecollagen matrix of claim 1, wherein the macroscopic collagen fibers havea length from about 3 to about 30 mm, from about 5 to about 25 mm orfrom about 7.5 to about 20 mm.
 3. A method for making a collagen matrixof claim 1, wherein the conditions sufficient to cause the collagenslurry to self-assembly comprise: (i) raising the pH of the slurry tofrom about 5 to about a pH of 9; or (ii) adding bone powder, calciumphosphate, hydroxyapatite, DBM or a mixture thereof to the slurry toraise the pH from about 5 to about
 9. 4. A method for making abiodegradable collagen matrix of claim 1, wherein the bioactive agent isuniformaly distributed throughout the collagen matrix.
 5. A method formaking a collagen matrix of claim 1, wherein the at least one bioactiveagent comprises a growth factor, a bone morphogenic protein, ananalgesic, an anti-inflammatory, and antibiotic, a cytokine, achemotherapeutic or a mixture thereof.
 6. A method for making a collagenmatrix of claim 5, wherein the at least one bone morphogenic protein isBMP-2.
 7. A method for making a collagen matrix of claim 2, furthercomprising drying the collagen matrix after it is formed.
 8. A method ofmaking a collagen matrix of claim 7, wherein the collagen matrix islyophilized prior to use.
 9. A method for making a collagen matrix ofclaim 8, further comprising adding glycerol to the bioactive agentbearing collagen matrix prior to lyophilizing the matrix.
 10. A methodfor making a collagen matrix of claim 1, wherein the collagen in thecollagen slurry comprises mammalian type I, type, II, type III, type IV,type IX, type X, type XI and type XII collagen, and mixtures thereof.11. A method for making a biodegradable collagen matrix of claim 1,wherein the collagen in the collagen slurry has not been treated forremoval of immunogenic protein.
 12. A method for making a collagenmatrix of claim 1, wherein the collagen matrix forms a moldablecomposition that does not harden.
 13. A method for making abiodegradable collagen matrix, the method comprising: adding a driedmixture of a bioactive agent and bone powder, calcium phosphate,hydroxyapatite, DBM or a mixture thereof to an acidic collagen slurry toform a collagen matrix of macroscopic collagen fibers wherein thebioactive agent is bound to the macroscopic collagen fibers.
 14. Amethod for making a biodegradable collagen matrix of claim 13, whereinthe bioactive agent is uniformly distributed throughout the collagenmatrix and the macroscopic collagen fibers have a length from about 3 toabout 30 mm, from about 5 to about 25 mm or from about 7.5 to about 20mm.
 15. A method for making a biodegradable collagen matrix of claim 13,further comprising removing water from the collagen matrix containingthe bioactive agent.
 16. A method for making a biodegradable collagenmatrix of claim 15, further comprising adding glycerol to the driedcollagen matrix to form a malleable complex that does not harden.
 17. Amethod for making a biodegradable collagen matrix of claim 13, whereinthe dried mixture is added to the acidic collagen slurry prior to use bya medical professional.
 18. A biodegradable collagen matrix for deliveryof a bioactive agent, the collagen matrix comprising a plurality ofmacroscopic collagen fibers comprising the bioactive agent bound to themacroscopic collagen fibers, the collagen matrix precipitated from anacidic collagen slurry by causing the pH of the slurry to be raisedabove a pH from about 5 to about a pH of
 9. 19. A biodegradable collagenmatrix for delivery of the bioactive agent of claim 18, wherein thebioactive agent comprises a growth factor, a bone morphogenetic protein,an analgesic, an anti-inflammatory, and antibiotic, a cytokine, achemotherapeutic or a mixture thereof.
 20. A biodegradable collagenmatrix for delivery of the bioactive agent of claim 19, wherein the bonemorphogenetic protein is BMP-2.